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| United States Patent |
5,550,213
|
|
Anderson
, et al.
|
August 27, 1996
|
Inhibitors of urokinase plasminogen activator
Abstract
A low molecular weight, reversible, proteinaceous inhibitor specific for
urokinase plasminogen activator is disclosed. Methods for designing,
constructing and using this and other such specific inhibitors of
urokinase plasminogen activator are also disclosed.
| Inventors:
|
Anderson; Stephen (Princeton, NJ);
Ryan; Raymond (Trenton, NJ)
|
| Assignee:
|
Rutgers, The State University of New Jersey (Piscataway, NJ)
|
| Appl. No.:
|
173102 |
| Filed:
|
December 27, 1993 |
| Current U.S. Class: |
530/324; 530/300 |
| Intern'l Class: |
C07K 014/00; C07K 014/465; A61K 038/00; A61K 038/57 |
| Field of Search: |
514/2
530/300
435/190
|
References Cited
U.S. Patent Documents
| 4258030 | Mar., 1981 | Sasaki et al. | 424/94.
|
| 4851345 | Jul., 1989 | Hayashi et al. | 435/215.
|
| 4952512 | Aug., 1990 | Lostukoff | 435/320.
|
| 5004609 | Apr., 1991 | Hayashi et al. | 424/94.
|
| 5098840 | Mar., 1992 | Kasai et al. | 435/215.
|
| Foreign Patent Documents |
| WO88/05081 | Jul., 1988 | WO.
| |
Other References
Fujinaga et al J. Mol. Biol. 195 pp. 397-418 (1987).
Turpeinen et al Biochem J. 254 pp. 911-914 (1980).
Schellenberger, V., et al., "Mapping the S' Subsites of Serine Proteases
Using Acyl Transfer to Mixtures of Peptide Nucleophiles," Biochemistry
Soc. 32: 4349-4353 (1993).
Hedstrom, L., et al., "Converting Trypsin to Chymotrypsin: The Role of
Surface Loops," Science 255:1249-1253 (1992).
Laskowski, Jr., M., et al., "Design of Highly Specific Inhibitors of Serine
Proteinases" Abstract: Protein Recognition of Immobilized Ligands, Alan R.
Liss, Inc. pp. 149-168 (1989).
Madison, E. L. et al., "Serpin-Resistant Mutants of Human Tissue-Type
Plasminogen Activator," Nature 339:721-724 (1989).
Blasi, F. et al., "Urokinase-Type Plasminogen Activator: Proenzyme,
Receptor, and Inhibitors," J. Cell. Biol. 104:801-804 (1987).
Carrell, R. W. et al., "Mobile Reactive Centre of Serpins and the Control
of Thrombosis," Nature 353:576-578 (1991).
Yu, H. et al., "Relationship between Secreted Urokinase Plasminogen
Activator Activity and Metastatic Potential in Murine B16 Cells
Transfected with Human Urokinase Sense and Antisense Genes," Canc. Res.
50:7623-7633 (1990).
Fujinaga, M. et al., "Crystal and Molecular Structures of the Complex of
.alpha.-Chymotrypsin with its Inhibitor Turkey Ovomucoid Third Domain at
1.8 .ANG. Resolution," J. Molec. Biol. 195:397 (1987).
|
Primary Examiner: Wax; Robert A.
Assistant Examiner: Moore; William W.
Attorney, Agent or Firm: Howrey & Simon, Auerbach; Jeffrey T.
Claims
What is claimed is:
1. A low molecular weight proteinaceous inhibitor of urokinase, wherein
said inhibitor is selected from the group consisting of:
(A) an inhibitor comprising an amino acid sequence of:
Val-Asp-Cys-Ser-Glu-Tyr-Pro-Lys-Pro-Ala-Cys-Gly-Arg-Thr-Gly-His-Pro-Leu-Cy
s-Gly-Ser-Asp-Asn-Lys-Thr-Tyr-Gly-Asn-Lys-Cys-Asn-Phe-Cys-Asn-Ala-Val
-Val-Flu-Ser-Asn-Gly-Thr-Leu-Thr-Leu-Ser-His-Phe-Gly-Lys-Cys (SEQ ID
NO:7);
(B) an inhibitor comprising an amino acid sequence of:
Val-Asp-Cys-Ser-Glu-Tyr-Pro-Lys-Pro-Ala-Cys-Ala-Arg-Met-Ala
-Ala-Pro-Leu-Cys-Gly-Ser-Asp-Asn-Lys-Thr-Tyr-Gly-Asn-Lys-Cys
-Asn-Phe-Cys-Asn-Ala-Val-Val-Glu-Ser-Asn-Gly-Thr-Leu-Thr-Leu
-Ser-His-Phe-Gly-Lys-Cys (SEQ ID NO:8);
(C) an inhibitor comprising an amino acid sequence of:
Val-Asp-Cys-Ser-Glu-Tyr-Pro-Lys-Pro-Ala-Cys-Gly-Arg-Val-Val
-Gly-Pro-Leu-Cys-Gly-Ser-Asp-Asn-Lys-Thr-Tyr-Gly-Asn-Lys-Cys
-Asn-Phe-Cys-Asn-Ala-Val-Val-Glu-Ser-Asn-Gly-Thr-Leu-Thr-Leu
-Ser-His-Phe-Gly-Lys-Cys (SEQ ID NO:9); and
(D) an inhibitor comprising an amino acid sequence of:
Val-Asp-Cys-Ser-Glu-Tyr-Pro-Lys-Pro-Ala-Cys-Ala-Arg-Ser-Ser
-Ala-Pro-Leu-Cys-Gly-Ser-Asp-Asn-Lys-Thr-Tyr-Gly-Asn-Lys-Cys
-Asn-Phe-Cys-Asn-Ala-Val-Val-Glu-Ser-Asn-Gly-Thr-Leu-Thr-Leu
-Ser-His-Phe-Gly-Lys-Cys (SEQ ID NO:10).
2. The inhibitor of claim 1 that comprises an amino acid sequence of (SEQ
ID NO:7):
Val-Asp-Cys-Ser-Glu-Tyr-Pro-Lys-Pro-Ala-Cys-Gly-Arg-Thr-Gly
-His-Pro-Leu-Cys-Gly-Ser-Asp-Asn-Lys-Thr-Tyr-Gly-Asn-Lys-Cys
-Asn-Phe-Cys-Asn-Ala-Val-Val-Glu-Ser-Asn-Gly-Thr-Leu-Thr-Leu
-Ser-His-Phe-Gly-Lys-Cys.
3. The inhibitor of claim 1 that comprises an amino acid sequence of (SEQ
ID NO:8):
Val-Asp-Cys-Ser-Glu-Tyr-Pro-Lys-Pro-Ala-Cys-Ala-Arg-Met-Ala
-Ala-Pro-Leu-Cys-Gly-Ser-Asp-Asn-Lys-Thr-Tyr-Gly-Asn-Lys-Cys
-Asn-Phe-Cys-Asn-Ala-Val-Val-Glu-Ser-Asn-Gly-Thr-Leu-Thr-Leu
-Ser-His-Phe-Gly-Lys-Cys.
4. The inhibitor of claim 1 that comprises an amino acid sequence of (SEQ
ID NO:9):
Val-Asp-Cys-Ser-Glu-Tyr-Pro-Lys-Pro-Ala-Cys-Gly-Arg-Val-Val
-Gly-Pro-Leu-Cys-Gly-Ser-Asp-Asn-Lys-Thr-Tyr-Gly-Asn-Lys-Cys
-Asn-Phe-Cys-Asn-Ala-Val-Val-Glu-Ser-Asn-Gly-Thr-Leu-Thr-Leu
-Ser-His-Phe-Gly-Lys-Cys.
5. The inhibitor of claim 1 that comprises an amino acid sequence of (SEQ
ID NO:10):
Val-Asp-Cys-Ser-Glu-Tyr-Pro-Lys-Pro-Ala-Cys-Ala-Arg-Ser-Ser
-Ala-Pro-Leu-Cys-Gly-Ser-Asp-Asn-Lys-Thr-Tyr-Gly-Asn-Lys-Cys
-Asn-Phe-Cys-Asn-Ala-Val-Val-Glu-Ser-Asn-Gly-Thr-Leu-Thr-Leu
-Ser-His-Phe-Gly-Lys-Cys.
6. The inhibitor of claim 1, wherein said inhibitor is joined to another
moiety.
7. The inhibitor of claim 6, wherein said moiety is a protein or a domain
of a protein.
8. The inhibitor of claim 6, wherein said joining is accomplished by
conjugating said inhibitor to said moiety.
9. The inhibitor of claim 8, wherein said moiety is a protein or a domain
of a protein.
10. The inhibitor of claim 7, wherein said joining is accomplished by
forming a fusion protein containing said inhibitor and said one or more
domains of a protein.
11. The inhibitor of claim 7, wherein said moiety comprises an antigen
binding domain of an antibody.
12. The inhibitor of claim 7, wherein said moiety comprises a receptor
ligand.
13. The inhibitor of claim 7, wherein said receptor ligand is a urokinase
receptor ligand.
14. The inhibitor of claim 7, wherein said moiety comprises a ligand
binding protein, or a domain thereof.
15. A low molecular weight proteinaceous inhibitor of urokinase, wherein
said inhibitor is produced by a process comprising the steps:
(A) identifying a set of amino acid residues in a proteinaceous urokinase
substrate that correspond to a set of amino acid residues in a Kazal-type
protease inhibitor by aligning:
(I) the amino acid sequence of said proteinaceous urokinase substrate, said
amino acid sequence (I) comprising a scissile bond-containing region of
said substrate,
with
(11) the amino acid sequence of the P1 loop of a Kazal-type protease
inhibitor, said amino acid sequence (11) comprising a P1 loop, and having
P2-P3' residues; such that the scissile bond of said substrate is aligned
with the P1-P1' bond of said protease inhibitor; wherein said aligned
amino acid sequences of (I) and (11) define said set of corresponding
amino acid residues; and
(B) synthesizing a proteinacous molecule wherein said P2-P3' residues of
said inhibitor have been replaced by the corresponding residues of said
substrate wherein said low molecular weight proteinaceous inhibitor of
urokinase comprises a polypeptide having an amino acid sequence selected
from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and
SEQ ID NO:10.
16. A low molecular weight proteinaceous inhibitor of urokinase, wherein
said inhibitor is produced by synthesizing a proteinacous molecule having
the amino acid sequence of a Kazal-type protease inhibitor, said inhibitor
having a P1 loop, and having P2-P3' residues, wherein said P2-P3' amino
acid residues of said inhibitor are amino acid residues of a proteinaceous
urokinase substrate that correspond to said P2-P3' amino acid residues
when:
(I) the amino acid sequence of said proteinaceous urokinase substrate, said
amino acid sequence (I) comprising a scissile bond-containing region of
said substrate,
is aligned with
(II) the amino acid sequence of the P1 loop of said Kazal-type protease
inhibitor, said amino acid sequence (II) comprising a P1 loop, and having
P2-P3' residues;
such that the scissile bond of said substrate is aligned with the P1-P1'
bond of said protease inhibitor.
Description
FIELD OF THE INVENTION
The invention relates to the design, construction and use of a low
molecular weight, reversible, proteinaceous inhibitor specific for
urokinase plasminogen activator.
BACKGROUND OF THE INVENTION:
I. Plasminogen and Plasminogen Activators
The serum protein, plasminogen, plays an integral role in the proteolytic
dissolution (or fibrinolysis) of blood clots. Plasminogen is an inactive
"proenzyme." It has a specific affinity for fibrin, and thus becomes
incorporated into blood clots as they form. Plasminogen's proteolytic
activity is released by "plasminogen activators" ("PA") that specifically
cleave the molecule to yield the active protease, plasmin. Plasmin is
capable of digesting the fibrin threads of blood clots, as well as other
substances involved in creating blood clots, such as fibrinogen, factor V,
factor VIII, prothrombin, and factor XII (for review, see Dan.o slashed.,
K. et al., Adv. Canc. Res. 44:139-266 (1985), herein incorporated by
reference)).
Plasmin is a serine protease, and exhibits substantial amino acid and
mechanistic homology with trypsin, chymotrypsin, and pancreatic elastase.
Plasmin has a relatively broad trypsin-like specificity, hydrolyzing
proteins and peptides at lysyl and arginyl bonds (Castellino, R. W. et
al., Meth. Enzymol. 80:365-380 (1981); Dane, K. et al., Adv. Canc. Res.
44:139-266 (1985)).
Agents that are capable of activating plasminogen (i.e. converting it to
plasmin) have been extensively studied. Two classes of natural mammalian
plasminogen activators have been described: urokinase-type plasminogen
activator and tissue-type plasminogen activator (Dane, K. et al., Adv.
Canc. Res. 44:139-266 (1985); Devlin, et al., PCT appl. WO88/05081; Kasaia
et al., U.S. Pat. No. 5,098,840; Hayashi, S. et al., U.S. Pat. No.
4,851,345; Sasaki et al., U.S. Pat. No. 4,258,030; Hayashi, S. et al.,
U.S. Pat. No. 5,004,609; Pyke, C. et al., Amer. J. Pathol. 138:1059-1067
(1991); Madison, E. L. et al., Nature 339:721-724 (1989); Blasi, F. et
al., J. Cell. Biol. 104:801-804 (1987)). These two classes of molecules
can be distinguished immunologically, by tissue localization, and by the
stimulation of their activity by fibrin. In addition, a third plasminogen
activator, streptokinase, has also been described. Streptokinase differs
from urokinase and tPA in that it is a bacterial protein produced by the
streptococci.
Urokinase-type plasminogen activator (UK) is a multi-domain protein with
one domain being a trypsin-like serine protease (Castellino, R. W. et al.,
Meth. Enzymol. 80:365-380 (1981); Dane, K. et al., Adv. Canc. Res.
44:139-266 (1985); Stra.beta.burger, W. et al., FEBS Lett. 157:219-223
(1983)). This protease domain converts plasminogen to plasmin by cleavage
at an arginyl residue (Castellino, R. W. et al., Meth. Enzymol. 80:365-380
(1981); Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985)). The
amino acid sequence and three-dimensional structure of several serine
proteases, including trypsin, chymotrypsin, and elastase have been deduced
(Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985);
Stra.beta.burger, W. et al., FEBS Lett. 157:219-223 (1983)).
Urokinase is synthesized in the kidneys, and can be recovered from urine.
It is initially produced as a single chain protein, "pro-urokinase" that
can be proteolytically cleaved by plasmin into an active two-chain protein
(Devlin, et al., PCT appl. WO88/05081).
Tissue-type plasminogen activator (tPA) is produced by the cells that line
the lumen of blood vessels or endothelial cells. Like urokinase, tPA is
also initially produced as a single-chain molecule (Rijken, D. G. et al.,
J. Biol. Chem. 256:7035-7041 (1981); Pennica, D. et al., Nature
301:214-221 (1983)).
The known plasminogen activators differ significantly in characteristics
such as their biological half-lives and their preference for fibrin. All
three classes of activators have been widely used as thrombolytic agents
for the treatment of thrombosis in myocardial infarction, stroke, arterial
occlusion, etc. (Kasai et al., U.S. Pat. No. 5,098,840; Hayashi et al.,
U.S. Pat. No. 5,004,609; Hayashi et al., U.S. Pat. No. 4,851,345; Sasaki
et al., U.S. Pat. No. 4,258,030).
II. Inhibitors of Plasminogen Activators
The regulation of fibrinolysis is crucial to the normal functioning of the
circulatory system. Thus, the activity of serum proteases (such as
urokinase or tPA) capable of activating plasminogen must be carefully
regulated to ensure that clot formation and dissolution can occur. One
manner in which such control is mediated concerns the regulated synthesis
of inhibitors of plasminogen activators.
Three classes of naturally occurring physiological inhibitors of the
plasminogen activators have been identified: the endothelial cell type
PA-inhibitor (PAI-1), the placental type PA-inhibitor (PAI-2), and
protease nexin-I (Sprengers, E. D. et al., Blood 69:381-387 (1987); Blasi,
F. et al., J. Cell Biol. 104:801-804 (1987); Madison, E. L. et al., Nature
339:721-724 (1989); Carrell, R. W. et al., Nature 353:576-578 (1991);
Lostukoff et al., U.S. Pat. No. 4,952,512; all herein incorporated by
reference). Such inhibitors comprise nearly 10% of the total protein in
blood plasma. They control a variety of critical events associated with
connective tissue turnover, coagulation, fibrinolysis, complement
activation and inflammatory reactions. Their function in the regulation of
the fibrinolytic system has not yet been fully clarified.
PAI-1 and PAI-2 have an approximate molecular weight of 50,000. They differ
in immunological reactivity and in their physiological characteristics
(Blasi, F. et al., J. Cell Biol. 104:801-804 (1987)). Whereas these
inhibitors are specific for PA, the protease nexin I inhibits plasmin,
thrombin, and other trypsin-like serine proteases in addition to PA
(Blasi, F. et al., J. Cell Biol. 104:801-804 (1987)).
PAI-1 is synthesized by a wide variety of cell types, including endothelial
cells, hepatocytes, several hepatoma cell lines, granulosa cells, and
possibly smooth muscle cells (Sprengers, E. D. et al., Blood 69:381-387
(1987); Blasi, F. et al., J. Cell Biol. 104:801-804 (1987)). PAI-1
interacts with both urokinase and tPA to form a stable bimolecular complex
that inactivates both the inhibitor and the PA (Kruithof, C.K.O. et al.,
Thromb.-Haemost. 55:65-68 (1986)). PAI-2 was first identified in placental
tissue. The inhibitor has been purified to homogeneity and found to have a
molecular weight of 48,000. It is not generally present in serum samples
(Sprengers, E. D. et al., Blood 69:381-387 (1987)). Protease nexin I is a
43,000 protein that exhibits a broad activity against trypsin-like serine
proteases. It is synthesized by fibroblasts, heart muscle cells, and
kidney epithelial cells (Sprengers, E. D. et al., Blood 69:381-387
(1987)).
III. The Role of Plasminogen Activators in Cancer
Metastasis involves the escape of a tumor cell from the tumor, its
translation to a new site, and its successful invasion of the tissue of
the new site and vascularization there to create a new tumor locus. The
membranes of vascular or lymphatic vessels and dense connective tissue
pose natural obstacles to the metastasis of tumor cells. The observation
that explants of cancer tissue consistently caused proteolytic degradation
eventually led to the recognition that tumor cells released a plasminogen
activator capable of converting plasminogen to plasmin (see, for review,
Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985)). By
secreting such a plasminogen activator, tumor cells are able to initiate a
cascade of reactions that results in the localized proteolysis needed for
tumor cell dissemination (Ossowski, L., Cell 52:321-328 (1988); Yu, H. et
al., Canc. Res. 50:7623-7633 (1990)).
Urokinase has been found to be secreted by a variety of tumor types,
including lung, colon and breast (see, Dan.o slashed., K. et al., Adv.
Canc. Res. 44:139-266 (1985) for review). Urokinase has been found to have
an important role in the metastasis of tumor cells (Yu, H. et al., Canc.
Res. 50:7623-7633 (1990); Ossowski, L., Cell 52:321-328 (1988); Ossoswki,
L. et al., Canc. Res. 51:274-281 (1991)). A positive correlation has been
reported between the metastatic potential of tumor cells and their
capacity to produce urokinase (Axelrod, J. H. et al., Molec. Cell. Biol.
9:2133-2141 (1989); Yu, H. et al., Canc. Res. 50:7623-7633 (1990)).
Significantly, urokinase appears to mediate its effect at the initial stage
of metastasis by facilitating the escape of tumor cells from the primary
tumor site. Indeed, inhibitors of urokinase, tested in a metastatic mouse
model, were ineffective in preventing either the translation or invasion
of of metastatic cells that had been injected into the animal's
bloodstream (Ostrowski, L. E. et al., Canc. Res. 46:4121-4128 (1986)).
Thus, UK inhibitors would be an especially desirable inhibitor of tumor
metastasis because they would not allow even the first stage of metastasis
to occur (i.e. escape of cells from the primary tumor site).
As indicated, urokinase is also produced in response to many natural
physiological conditions. The implantation of a fertilized egg into the
uterine wall provides an example of localized proteolysis, mediated by
urokinase, that occurs in normal tissue (Dane, K. et al., Adv. Canc. Res.
44:139-266 (1985)).
In view of the role of plasminogen activators, in general, and of
urokinase, in particular, in the metastasis of tumor cells, and in
mediating uterine implantation, it would be desirable to have a low
molecular weight, reversible, proteinaceous inhibitor specific for
urokinase that could be employed in the treatment of metastatic cancers,
or in the prevention of pregnancy. No low molecular weight, protein
inhibitors with significant functional affinity for UK have been
previously identified. The present invention provides such molecules, and
methods for their use.
SUMMARY OF THE INVENTION
Urokinase plasminogen activator (UK) is a multi-domain protein with one
domain being a serine protease showing sequence homology to the trypsin
family of serine proteases. This protease domain converts plasminogen to
plasmin. The present invention relates to the design, construction, and
use of low molecular weight, reversible, proteinaceous inhibitors specific
for urokinase plasminogen activator.
In detail, the invention provides a low molecular weight proteinaceous
inhibitor of urokinase, and, in particular, a Kazal-type inhibitor of
urokinase. The invention particularly provides Kazal-type inhibitors of
urokinase that are related to human pancreatic secretory trypsin inhibitor
and/or an avian ovomucoid third domain.
The invention further provides Kazal-type inhibitors of urokinase that have
mutations in residues of an avian ovomucoid third domain.
The invention further provides a low molecular weight inhibitor of
urokinase that is joined to another moiety, such as an antibody, a ligand
for a receptor molecule, or a receptor molecule.
In particular, the invention provides low molecular weight proteinaceous
inhibitors of urokinase having the amino acid sequence:
______________________________________
Val--Asp--Cys--Ser--Glu--Tyr--Pro--Lys--Pro--Ala--
Cys--Gly--Arg--Thr--Gly--His--Pro--Leu--Cys--Gly--
Ser--Asp--Asn--Lys--Thr--Tyr--Gly--Asn--Lys--Cys--
Asn--Phe--Cys--Asn--Ala--Val--Val--Glu--Ser--Asn--
Gly--Thr--Leu--Thr--Leu--Ser--His--Phe--Gly--Lys--
Cys;
or
Val--Asp--Cys--Ser--Glu--Tyr--Pro--Lys--Pro--Ala--
Cys--Ala--Arg--Met--Ala--Ala--Pro--Leu--Cys--Gly--
Ser--Asp--Asn--Lys--Thr--Tyr--Gly--Asn--Lys--Cys--
Asn--Phe--Cys--Asn--Ala--Val--Val--Glu--Ser--Asn--
Gly--Thr--Leu--Thr--Leu--Ser--His--Phe--Gly--Lys--
Cys;
or
Val--Asp--Cys--Ser--Glu--Tyr--Pro--Lys--Pro--Ala--
Cys--Gly--Arg--Val--Val--Gly--Pro--Leu--Cys--Gly--
Ser--Asp--Asn--Lys--Thr--Tyr--Gly--Asn--Lys--Cys--
Asn--Phe--Cys--Asn--Ala--Val--Val--Glu--Ser--Asn--
Gly--Thr--Leu--Thr--Leu--Ser--His--Phe--Gly--Lys--
Cys;
or
Val--Asp--Cys--Ser--Glu--Tyr--Pro--Lys--Pro--Ala--
Cys--Ala--Arg--Ser--Ser--Ala--Pro--Leu--Cys--Gly--
Ser--Asp--Asn--Lys--Thr--Tyr--Gly--Asn--Lys--Cys--
Asn--Phe--Cys--Asn--Ala--Val--Val--Glu--Ser--Asn--
Gly--Thr--Leu--Thr--Leu--Ser--His--Phe--Gly--Lys--
Cys.
______________________________________
The invention also provides a DNA molecule that encodes a low molecular
weight proteinaceous inhibitor of urokinase, and more specifically each of
the above recited inhibitors.
The invention also provides a method of preventing or attenuating a
urokinase-dependent process (such as the metastasis of a tumor, ovulation,
uterine implantation of a fertilized ova, etc.) in an individual which
comprises administering to the individual an effective amount of a low
molecular weight proteinaceous inhibitor of urokinase.
The invention also provides an antineoplastic agent comprising a low
molecular weight proteinaceous inhibitor of urokinase.
The invention further provides a contraceptive agent comprising a low
molecular weight proteinaceous inhibitor of urokinase.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is an illustration of the canonical loop by a superimposition of the
backbone nitrogen, .alpha.-carbons, carbonyl carbons, and side chain
.beta.-carbons, with an RMS deviation of 0.2 .ANG.. The OM3TKY (labelled
PDB1CHO), and BPTI (labelled PDB1TPA), from residue P6 to residue P6' are
shown. The catalytic triad in relation to the P1 loop is also shown.
FIG. 2 shows plots of initial crude assays to determine inhibitory activity
of inhibitor constructs. The numbers in the legends refer to the
oligonucleotide numbers. Absorbance at 405 nm and activity were determined
in a Kontron Uvikon 860 spectrophotometer. The top panel shows
chymotrypsin activity versus inhibitor concentration in .mu.M.
Chymotrypsin concentration was 0.1 .mu.M. The lower panel shows urokinase
activity versus inhibitor concentration in .mu.M. Urokinase concentration
was 1.6 .mu.M.
FIGS. 3A-3F show plots of inhibitor concentration (in nM) versus free
urokinase (in nM). The experimental data (open circles is plotted against
the theoretical curve (solid line) for an inhibitor for the given K.sub.i
value. The K.sub.i values were determined using Enzfitter (Elsevior
Biosoft). The data is presented for wild-type "WT" inhibitor (FIG. 3A) and
for five mutant inhibitors: "Arg18" (FIG. 3B), "88" (FIG. 3C), "89" (FIG.
3D), "90" (FIG. 3E) and "91" (FIG. 3F).
DESCRIPTION OF THE PREFERRED EMBODIMENTS:
The present invention concerns the design, construction, and use of low
molecular weight, reversible, proteinaceous inhibitors specific for
urokinase plasminogen activator. To accomplish this goal, the present
invention exploits homologies and similarities in structure between
urokinase and the trypsin-like serine proteases to model the serine
protease domain of UK. Such inhibitors can be designed by
computer-assisted modeling based upon the structure of urokinase, and its
similarity to other proteins.
Increasingly, the correlation between the structures of known proteins and
the sequence of a target protein is made using computer simulations (van
Gunsteren, V. F., Prot. Engin. 2:5-13 (1988); Yang, M. M. et al., In:
Reaction Centers of Photosynthetic Bacteria, (Michel-Beyerle, Ed.),
Springer-Verlag, Germany (1990), pp 209-218), databases (Moult, J. et al.,
Proteins 1:146-163 (1987); Klein, P. et al., Biopolymers 25:1659-1672
(1986); Nakashima, H. et al., J. Biochem. (Tokyo) 99:153-162 ((1986);
Deleage, G. et al., Prot. Engin. 1:289-294 (1987)); neural networks (Qian,
N. et al., J. Molec. Biol. 202:865-884 (1988); Holley, L. H. et al., Proc.
Natl. Acad. Sci. (U.S.A.) 86:152-156 (1989); Bohr, H. et al., FEBS Lett.
241:223-228 (1988)); or expert systems (Robson, B. et al., J. Molec.
Graphics 5:8-17 (1987)). See, generally, Fasman, G. R., TIBS 14:295-299
(1989)).
The use of computers or computer-assisted methods in analyzing the
structure of proteins is discussed, for example, in U.S. Pat. Nos.
4,704,692 (Ladner); 4,760,025 (Estell et al.); 4,853,871 (Pantoliano et
al.); and 4,908,773 (Pantoliano et al.).
Modeling information obtained for urokinase can be employed to define
inhibitors of tPA as well as for urokinase. UK and tPA are both
multi-domain proteins. Both have a serine protease domain and an EGF-like
domain. UK has one kringle domain, while t-PA has two such domains, as
well as a fibronectin-like finger domain (Dan.o slashed., K. et al., Adv.
Canc. Res. 44:139-266 (1985)). Both plasminogen activators cleave the same
substrate, plasminogen, at the same arginyl site, Arg 561 in plasminogen
(Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981)). Both
plasminogen activators have the same natural inhibitors (Sprengers, E. D.
et al., Blood 69:381-387 (1987)). The functional distinction between the
two plasminogen activators derives in part from the localization of the
two enzymes within an organism. This localization is a function of the
non-proteolytic domains (Behrendt, N. et al., J. Biol. Chem. 266:7842-7847
(1991); Blasi, F. et al., J. Cell Biol. 104:801-804 (1987); Dan.o
slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985)). Also, the
naturally occurring inhibitors to the plasminogen activators are
preferential in reactivity to UK or tPA, but not solely specific for
either one (Sprengers, E. D. et al., Blood 69:381-387 (1987)). Using UK as
a model system for comparison with tPA allows for a finer determination of
what governs recognition and specificity in the protease domains.
I. Trypsin-Like Serine Proteases
The trypsin-like serine proteases are hydrolases which utilize a catalytic
triad of amino acids made up of histidine, serine, and aspartic acid to
cleave a peptide substrate at the peptide bond between two residues of the
substrate, S1 and S1'. The nomenclature for the substrate residues derives
from their position relative to the scissile (S) peptide bond (Wynn, R.,
Ph.D. Dissertation, Purdue University, pp 28-30; 50-115 (1990)). This
numbering extends outward in either direction from the scissile bond,
which is the site of cleavage. Enzymology and kinetics studies, coupled
with crystallographically determined models of serine proteases, have
enabled researchers to describe in molecular detail the mechanism of
serine-protease catalysis (Wynn, R., Ph.D. Dissertation, Purdue
University, pp 28-30; 50-115 (1990)). The three-dimensional structures of
several trypsin-like serine proteases show that the structural homology of
this enzyme family extends beyond primary structure to the
three-dimensional structures (Fujinaga, M. et al., J. Molec. Biol. 195:397
(1987); Huber, R. et al., J. Molec. Biol. 89:73 (1974); Stra.beta.burger,
W. et al., FEBS Lett. 157:219-223 (1983)). These structural similarities
have helped to emphasize the structural and functional relationships of
the catalytic mechanism, but the details of the specificity mechanisms of
these proteases are still unclear.
The differences among the homologous protease structures have also been
used to understand the functional differences in substrate specificity
between many members of the trypsin-like serine protease family. While the
mechanism of proteolysis is the same for these enzymes, the specificity of
the enzymes is quite varied (Table 1).
TABLE 1
______________________________________
TRYPSIN-LIKE SERINE PROTEASES
______________________________________
Protease S1 Specificity
Trypsin Arg, Lys
Chymotrypsin Phe, Tyr, Trp
Elastase Leu, Val, Ala
Thrombin Arg, Lys
Plasminogen Arg, Lys
tPA, UK Arg, Lys
______________________________________
The cleavage specificity of a trypsin-like serine protease is thought to
rely primarily on residues in the active site pocket, which interact with
the S1 residue of the substrate (Chase, T. et al., Biochem. Biophys. Res.
Commun. 29:508-514 (1967); Hedstrom, L. et al., Science 255:1249-1253
(1992); Wynn, R., Ph.D. Dissertation, Purdue University, pp 28-30; 50-115
(1990)). The S1 specificity for basic residues has been attributed to a
complementary charge of an aspartic acid at position 189
(trypsin/chymotrypsin numbering) (Hedstrom, L. et al., Science
255:1249-1253 (1992)), which lies at the bottom of the protease active
site. More recent studies have indicated that loops of the protease
outside the active site pocket help determine the substrate specificity
(Hedstrom, L. et al., Science 255:1249-1253 (1992)).
II. Proteinaceous Serine Protease Inhibitors
A structural understanding of the specificity of the trypsin-like proteases
can be achieved through chemical and crystallographic studies of various
enzymes in complex with protease inhibitors, synthetic and proteinaceous.
As indicated in Table 2, there are many families of proteinaceous serine
protease inhibitors (Onesti, S. et al., J. Molec. Biol. 217:153-176
(1991); Wynn, R., Ph.D. Dissertation, Purdue University, pp 28-30; 50-115
(1990)).
TABLE 2
______________________________________
PROTEIN SERINE PROTEASE INHIBITORS
Animals Plants Microbial
______________________________________
Kunitz Soybean Trypsin (Kunitz)
SSI
(e.g., BPTI)
Kazal Soybean (Bowman-Birk)
(e.g., PSTI)
Ascaris Potato 1
Hirudin Barley Trypsin
Serpin Squash
______________________________________
Most of the above-listed inhibitors act reversibly. The serpin (SERine
Protease INhibitor) family of inhibitors, however, act irreversibly
(Carrell, R. W. et al., Nature 353:576-578 (1991); Sprengers, E. D. et
al., Blood 69:381-387 (1987)). Urokinase's two known native inhibitors,
PAI-1 and PAI-2 (Sprengers, E. D. et al., Blood 69:381-387 (1987)) are
members of the serpin family of inhibitors (Christensen, U. et al.,
Thromb.-Haemost. 48:24-26 (1982); Kruithof, C.K.O. et al.,
Thromb.-Haemost. 55:65-68 (1986); Sprengers, E. D. et al., Blood
69:381-387 (1987)). The structure of these serpin inhibitors when
complexed with a protease is not known. Another serpin, protease nexin-1,
has also been seen to have some inhibitory effect against UK, though not
against tPA (Sprengers, E. D. et al., Blood 69:381-387 (1987)).
The sizes of these proteinaceous inhibitors vary greatly, ranging from
approximately 5 kD for the squash inhibitor to about 60 kD for the
serpins. The nomenclature for the residues of the inhibitors mimics that
for protease substrates, using the labels P1 and P1' in place of S1 and
S1' (Wynn, R., Ph.D. Dissertation, Purdue University, pp 28-30; 50-115
(1990)).
The crystal and NMR structures of several inhibitor-protease complexes have
been determined (Bode, W. et al., Eur. J. biochem. 147:387-395 (1985);
Fujinaga, M. et al., J. Molec. Biol. 195:397 (1987); Huber, R. et al., J.
Molec. Biol. 89:73 (1974); Papamokos, E. et al., J. Molec. Biol.
158:515-537 (1982)).
An interesting feature common to these complexed structures is the
canonical active site loop structure of the inhibitor (Bode, W. et al.,
Eur. J. biochem. 147:387-395 (1985); Carrell, R. W. et al., Nature
353:576-578 (1991); Onesti, S. et al., J. Molec. Biol. 217:153-176 (1991);
Wynn, R., Ph.D. Dissertation, Purdue University, pp 28-30; 50-115 (1990)).
This canonical loop is the inhibitor loop that binds the protease active
site pocket; P1 and P1' appear the same distance apart as they do in the
non-complexed inhibitor. Conformational similarity of the P1 loop between
quite different inhibitors, particularly in the backbone conformation, has
been remarked upon (Bode, W. et al., Eur. J. biochem. 147:387-395 (1985);
Onesti, S. et al., J. Molec. Biol. 217:153-176 (1991); Wynn, R., Ph.D.
Dissertation, Purdue University, pp 28-30; 50-115 (1990)).
The serpins are, however, an exception to these observations. No crystal
structures have been determined for inhibitory serpins in either their
native (uncleaved) state, or in complex with a protease. Those crystal
structures that do exist are of either the non-inhibitory serpin-like
protein, ovalbumin, or they are of the stable form of inhibitory serpins,
cleaved at the P1-P1' peptide bond (Carrell, R. W. et al., Nature
353:576-578 (1991); Stein, P. E. et al., Nature 347:99-102 (1990)). The
serpin structures do not exhibit the canonical inhibitor loop seen in
other inhibitor-protease complexes. The non-inhibitory ovalbumin has an
alpha helix corresponding to the active site loop (Stein, P. E. et al.,
Nature 347:99-102 (1990)), and in the structures of the cleaved inhibitory
serpins, the P1 and P1' residues are almost 70A apart from each other
(Carrell, R. W. et al., Nature 353:576-578 (1991)).
III. Production of Urokinase Inhibitors
A. Identification of Urokinase Inhibitors
Due to the lack of structural knowledge of serpins complexed with
proteases, the small, protein protease inhibitors were used as a model
system for protein recognition.
In the initial step in the modelling, the amino acid sequences of urokinase
(UK), tissue plasminogen activator (tPA), bovine chymotrypsinogen (CHYM),
bovine trypsin (TRYP) and porcine elastase (ELASTASE) serine protease
domains were aligned. For this purpose, the protein sequence alignment of
these proteins determined by Stra.beta.burger, W. et al., FEBS Lett.
157:219-223 (1983), herein incorporated by reference, was used.
The UK active site and surrounding structure of UK was modelled using a
structure of trypsin complexed with bovine pancreatic trypsin inhibitor,
BPTI (Brookhaven file 1TPA (Huber, R. et al., J. Molec. Biol. 89:73
(1974)) as a starting framework. The modelling was done using Insight,
Version 2.5 (Biosym), although other suitable programs or methods could
also have been employed. For such modeling, a series of amino acid
substitutions were made in the trypsin structure so as to introduce the
corresponding residues present in UK. To simplify the modeling of UK, the
enzymes' surface residues closest to the active site pocket, judged as
being important for a working model, were preferentially replaced.
In order to maximize the alignment of the amino acid sequences of trypsin
and urokinase, three "loops" are preferably created in the urokinase
sequence to account for the presence of amino acid residues in urokinase
that lack a counterpart in trypsin (i.e. possess a "gap" when properly
aligned). Thus, in order to adapt the structure of trypsin to form a model
for urokinase, it was desirable to add a series of three insertions at the
"loop" points in the trypsin structure.
The present invention follows the convention of Stra.beta.burger, W. et al.
(FEBS Lett. 157:219-223 (1983)) in referring to the residues of the
aligned molecules with reference to the corresponding residue number of
chymotrypsinogen. Thus for example, residue 35 of trypsin is that residue
of trypsin which corresponds in location to residue 35 of chymotrypsinogen
when the two molecules are aligned to maximize homology.
The first insertion was intended to "fill" the "gap" at residues 35 through
38 of trypsin. Residues 28-42 of elastase (Brookhaven files 1EST (Sawyer,
L. et al,, J. Molec. Biol. 118:137 (1978))) were selected to comprise the
first loop insertion. Thus, these residues replaced the region of trypsin
in the model that corresponded in alignment to chymotrypsinogen residues
28-42. The one amino acid corresponding to urokinase that was still
lacking (i.e. His 36) was inserted singly into the break between residues
34 and 35 of elastase.
The second insertion was based on a loop originally derived from the
Brookhaven file 1CAC, and selected by an internal geometry search of the
Protein Database. Backbone torsion angles were checked by the Ramachandran
function of TOM (FRODO for the IRIS SG). Amino acid substitutions were
made in these insertions to obtain UK sequences.
The third insertion was also taken from elastase, and covered the "gap"
between residues 99 and 100 of trypsin. This insertion actually replaced
trypsin residues from 90 to 105 with the corresponding residues in
elastase.
Other gaps in the alignment of trypsin and urokinase were deemed to be
sufficiently far from the inhibitor binding determinants as to not play a
vital role in the protease-inhibitor interaction. Accordingly, they were
not "corrected" in the model.
In order to identify a suitable small inhibitor of urokinase, the complexed
structures of known proteases and their inhibitors were considered. Among
such protease inhibitors whose protease complex structures are known are
Bovine Pancreatic Trypsin Inhibitor (BPTI), a Kunitz inhibitor (Huber, R.
et al., J. Molec. Biol. 89:73 (1974); Wynn, R., Ph.D. Dissertation, Purdue
University, pp 28-30; 50-115 (1990)), and some Avian Ovomucoid Third
Domain (OM3) variants, which are Kazal-type inhibitors (Fujinaga, M. et
al., J. Molec. Biol. 195:397 (1987); Papamokos, E. et al., J. Molec. Biol.
158:515-537 (1982), herein incorporated by reference). A third variety of
proteinaceous protease inhibitors, from the soybean E. latissima, may also
be used to design an inhibitor for plasminogen activator (Onesti, S. et
al., J. Molec. Biol. 217:153-176 (1991)). Preferably, the desired
inhibitor of urokinase is designed by modifying small protease inhibitors
of well known structure, and most preferably, inhibitors from the
Kazal-type family.
The modeling program was therefore used to complex BPTI with the protease
"loops" surrounding the UK active site, thereby forming a UK-BPTI. Such
complexing did not necessarily sterically interfere with a small
inhibitor's placement in the active site pocket in this preliminary model.
Likewise, no overtly poor charge interactions resulted from having BPTI in
complex with the UK model. A "worst case" UK-BPTI model still only allowed
for four poor steric interactions of the inhibitor and protease. These
possible bad contacts were in region 37-42 of BPTI.
The UK-BPTI model was then compared with the structural model of
chymotrypsin complexed with a second protease inhibitor, the ovomucoid
third domain (OM3). A superimposition of the trypsin and chymotrypsin
structures, using homologous amino acids in the proteases, gave a low RMS
deviation for both the protease amino acids and the P1 loops of each
inhibitor, residues P3 to P3'. A second superimposition of the two
structures was performed using the alpha and beta carbons of P2 to P3' of
each inhibitor, and the protease catalytic triad. Even though the side
chains of each inhibitor were different from each other, the RMS deviation
of backbone and beta carbon configuration of the inhibitors was below 0.2
A deviations (FIG. 1). FIG. 1 illustrates the canonical loop.
The interactions that the two inhibitors, BPTI and OM3 might have with the
protease were evaluated using computer modeling. The BPTI P1 loop, though
narrower from P4-P3', was found to be less extended in comparison with the
P1 loop of OM3, and thus more discontinuous strands of BPTI could possibly
interact with the protease model.
Kazal-type inhibitors display a great deal of flexibility in the P1 loop
binding the protease active site, as evidenced by their ability to bind
both trypsin-like and subtilisin-like proteases with different P1 loop
conformations. BPTI is not known to have inhibitory effects against
subtilases. These factors suggested that changes within the ovomucoid
third domain (OM3) active site would cause fewer perturbations on the
surrounding inhibitor structure than would changes in BPTI. Moreover,
fewer mutations would be needed in order to make an OM3-derived inhibitor
with affinity for UK.
Thus, the above-described computer modelling indicated that a desired UK
inhibitor could be obtained via mutation of a Kazal-type inhibitor, and
specifically the turkey ovomucoid third domain (OM3TKY) Kazal-type
inhibitor.
By starting from OM3TKY, it was also possible to apply the results of many
structural and functional studies that have been done to investigate amino
acid substitutions within the OM3 framework (Empie, M. W. et al., Biochem.
21:2274-2284 (1982); Laskowski, M. et al., Biochem. 30:10832-10838 (1991);
Papamokos, E. et al., J. Molec. Biol. 158:515-537 (1982); Wynn, R., Ph.D.
Dissertation, Purdue University, pp 28-30; 50-115 (1990), all herein
incorporated by reference). Laskowski and co-workers have mapped and
summarized a list of primary and secondary contact sites of the OM3 domain
variants against a spectrum of trypsin-like proteases. The consensus
contact residues of OM3 domains is summarized in Table 3 (Laskowski, M. et
al., Biochem. 30:10832-10838 (1991); Wynn, R., Ph.D. Dissertation, Purdue
University, pp 28-30; 50-115 (1990)). In Table 3, primary contacts
(1.degree.) are defined by non-hydrogen atoms of the inhibitor that are
within 4 A of non-hydrogen atoms of the protease. An asterisk (*) denotes
a hydrogen bond interaction, while the dagger (.dagger.) denotes a residue
participating in an ion-binding pair.
TABLE 3
______________________________________
PROTEASE CONTACT RESIDUES OF OM3
Main Chain (MC) or
1.degree. or 2.degree.
Side Chain (SC) Structurally
Residue Contact Contact Conserved
______________________________________
P6 1.degree.
MC* N
P5 -- -- Y (Pro)
P4 1.degree.
SC N
P3 -- -- Y (Cys)
P2 1.degree.
SC* N
P1 1.degree.
SC, MC N
P1' 1.degree.
SC N
P2' 1.degree.
SC.dagger. N
P3' 1.degree.
-- N
P14' 2.degree.
-- Y (Gly)
P15' 2.degree.
SC* Y (Asn)
P18' 1.degree.
SC N
______________________________________
Those residues listed in Table 3 as structurally conserved are deemed
important to maintain the inhibitors' structures, and not for value of
their direct interaction with a protease (Wynn, R., Ph.D. Dissertation,
Purdue University, pp 28-30; 50-115 (1990)). Primary contacts (1.degree.)
designate residues in the inhibitor P1 loop, or those residues which make
direct contact with the protease. Secondary contacts (2.degree.) are those
residues of the inhibitor which interact indirectly via contacts with the
P1 loop. Residue P14' seems to be conserved as a glycine in most OM3
domains, but whether the affects of substitutions at this site are
intra-structural for the inhibitor, or contact determined between protease
and inhibitor, is unclear. Effects on free energy and disassociation
constants by substitutions at P14' have been shown to be non-additive
(Wynn, R., Ph.D. Dissertation, Purdue University, pp 28-30; 50-115
(1990)).
Although changes in conserved structural residues, such as P5, P3, and P14'
might yield strong inhibitors, such an alteration would potentially
obscure the structural basis for any observed change in inhibitor
specificity. Moreover, changes in any of the conserved structural residues
might alter the inhibitor structure non-predictably. For these reasons, no
changes were made at any conserved residues.
It is desirable to take advantage of the structurally conserved nature of
the P1 loop. Since the backbone and carbon structures remain relatively
constant, the nature of the side chains, whether basic, acidic, aliphatic,
aromatic, long, or short, is a major determining factor for inhibitor
affinity.
The knowledge of specificity determinants between tPA and PAI-1 (Madison,
E. L. et al., Nature 339:721-724 (1989)) was an additional consideration
in designing the desired inhibitor. tPA contains a loop corresponding to
the first insertion (discussed above) in the UK model. This loop governs
the interaction of tPA with PAI-1. This loop is located nearer the
C-terminal P' residues of postulated inhibitor interactions, rather than
on the N-terminal side of P1. The working model of UK in complex with
inhibitors did not show any interaction with this loop. Likewise, changes
in this loop did not seem to affect tPA activity against plasminogen,
suggesting a specificity determinant further from the P1 loop.
Because it was desired that the inhibitors identified by the present
invention would not simply have high affinity for UK, but would also have
higher specificity for UK, the effect of changes in residues in the P1
loop was considered as a means to achieve more than arginyl specificity
from P1. Specifically, changes were made in the P1 loop C-terminal to P1,
in the P1'-P3' region nearest the t-PA loop. The region N-terminal to P2
was not altered, due to the presence of more structurally conserved
residues in this part of the P1 loop.
As a result of the above modeling considerations, replacement of the short
P1 loop of the OM3TKY with the region immediately surrounding the scissile
bond of PAI-1, PAI-2, plasminogen, and Nexin-1 was considered a likely
means for obtaining a urokinase-specific inhibitor. Table 4 shows the
mutations identified as having potential for producing such an inhibitor.
The substituted residues are indicated with asterisks. The P1 residue,
leucine, of OM3TKY is also indicated with an asterisk. The cysteines are
in boldface, to indicate that they participate in disulfide bonds.
TABLE 4
__________________________________________________________________________
MUTATIONS IN OM3TKY IDENTIFIED AS RESULTING
IN A POTENTIAL UROKINASE INHIBITOR
PROTEASE INHIBITOR
RESIDUE
OM3TKY
Plasminogen Derived
PAI-2 Derived
PAI-1 Derived
Nexin Derived
__________________________________________________________________________
P5 P P P P P
P4 A A A A A
P3 C C C C C
P2 T G* G* A* A*
P1 L* R* R* R* R*
P1' E V* T* M* S*
P2' Y V* G* A* S*
P3' R G* H* A* A*
P4' P P P P P
P5' L L L L L
P6' C C C C C
P7' G G G G G
P8' S S S S S
P9' D D D D D
__________________________________________________________________________
B. Structural Considerations in the P1 Loop All of the proteins with
natural affinity for UK have a glycine or alanine at P2, as do all of the
proposed mutants. The presence of a glycine or alanine would allow a
greater deal of X-.PSI. torsional freedom than the .beta.-branched
threonine of WT OM3TKY. Additionally, the O.gamma. of the P2 Thr in OM3TKY
is known to hydrogen bond with the P1' residue backbone ((Wynn, R., Ph.D.
Dissertation, Purdue University, pp 28-30; 50-115 (1990)), possibly
conferring more rigidity on the P1 loop. The removal of this rigidity
could also increase the flexibility of the loop in possible mutants.
Other changes that could introduce greater loop flexibility are the
introduction of glycine and serine residues in the P1'-P3' residues of the
mutants. All of the potential mutants are bounded at P4' by the proline
which induces a turn in the loop back into the cystine scaffold of the
Kazal inhibitor. A Pro to Gly change at P4' has the potential to further
increase P1 loop flexibility.
C. Possible Effects of Secondary Contacts
The mutations formed by replacement of OM3TKY active residues by the serpin
and plasminogen residues leave a pocket between Asn 33 of OM3TKY and the
position formerly occupied by Glu 19 (P1' of OM3TKY) (Wynn, R., Ph.D.
Dissertation, Purdue University, pp 28-30; 50-115 (1990)). Asn 33 of
OM3TKY is the P15' residue. This pocket, roughly the size of a methyl
group might cause a slight collapse of the surrounding P1 backbone
conformation, especially due to the glycines and alanines substituted at
P2. The P15' Asn site has been seen as an important mediator of inhibitor
affinity in other studies with avian ovomucoid third domains (Empie, M. W.
et al., Biochem. 21:2274-2284 (1982); Laskowski, M. et al., Biochem.
30:10832-10838 (1991); Papamokos, E. et al., J. Molec. Biol. 158:515-537
(1982); Wynn, R., Ph.D. Dissertation, Purdue University, pp 28-30; 50-115
(1990)).
If one desired to prevent this pocket from permitting such a collapse to
occur, the P15' in OM3TKY could be changed to one of the following three
residues: glutamine, leucine, or isoleucine, in that order of preference.
In sum, the present invention uses a computer-assisted modeling method to
identify inhibitors of urokinase and other proteins of similar structure.
D. Synthesis of Inhibitor Molecules
The inhibitor molecules of the present invention may be made by chemical
synthesis, such as by peptide synthesis. More preferably, the inhibitor
molecules will be made using recombinant DNA technology. In this
embodiment, gene sequences capable of expressing the inhibitor, or a
fusion protein of the inhibitor with another protein, peptide or amino
acid leader or trailing sequence, will be introduced into a vector, such
as a cosmid, bacteriophage or plasmid and introduced into a suitable host
cell.
Any means known in the art may be used to synthesize the oligonucleotides
that encode the inhibitors of the present invention (Zamechik et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 83:4143 (1986); Goodchild et al., Proc.
Natl. Acad. Sci. (U.S.A.). 85:5507 (1988); Wickstrom et al., Proc. Natl.
Acad. Sci. (U.S.A.) 85:1028; Holt, J. T. et al., Mol. Cell. Biol. 8:963
(1988); Gerwirtz, A. M. et al., Science 242:1303 (1988); Anfossi, G., et
al., Proc. Natl. Acad. Sci. (U.S.A.) 86:3379 (1989); Becker, D., et al.,
EMBO J. 8:3679 (1989); all of which references are incorporated herein by
reference) . Automated nucleic acid synthesizers may be employed for this
purpose. In addition, desired nucleotides of any sequence can be obtained
from any commercial supplier of such custom molecules.
Most preferably, such oligonucleotides may be prepared using solid phase
"phosphoramidite synthesis." The synthesis is performed with the growing
nucleotide chain attached to a solid support derivatized with the
nucleotide which will be the 3'-hydroxyl end of the transcript or
oligonucleotide. The method involves the cyclical synthesis of DNA using
monomer units whose 5'-hydroxyl group is blocked (preferably with a 5'-DMT
(dimethoxytrityl) group), and whose amino groups are blocked with either a
benzoyl group (for the amino groups of cytosine and adenosine) or an
isobutyryl group (to protect guanosine). Methods for producing such
derivatives are well known in the art.
The DNA molecules thereby produced may be incorporated into "vector"
molecules. The term "vector," as used herein is intended to denote any
viral or plasmid molecule that is capable of being introduced (as by
transformation, electroporation, transfection, etc.) and/or propagated
(i.e. replicated) in a prokaryotic or eukaryotic cell. Suitable
prokaryotic or eukaryotic vectors, as well as the methods for using them
are disclosed, for example, by Sambrook, J. et al., In: Molecular Cloning
A Laboratory Manual, 2nd Edition, (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989), herein incorporated by reference).
For some purposes, such as the isolation of inhibitor in quantities
sufficient to facilitate in vitro purification, it may be desirable to
express the inhibitors of the present invention in prokaryotic hosts,
especially bacteria using prokaryotic vectors. Preferred prokaryotic
vectors include plasmids such as those capable of replication in E. coli
such as, for example, pBR322, pEZZ318, ColE1, pSC101, pACYC 184, .pi.VX.
Such plasmids are, for example, disclosed by Sambrook, J. et al. (In:
Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. (1989 ) ) .
A preferred prokaryotic vector is one that mediates the expression of the
protein as a fusion protein with staphylococcal protein A ("SPA").
Examples of such vectors include the pRIT 20 and pRIT 30 series of vectors
(Nilsson, B. et al. (Meth. Enzymol. 185:144-161 (1990)). Because SpA
fusions are expressed in diverse bacterial hosts, and direct the fusion
protein into either the periplasmic space or the culture medium, such
fusions facilitate the purification of the protein. Moreover, since SpA
binds to the Fc portion of IgG, the fusion protein can be readily purified
by immunoaffinity methods. The construction and use of such SpA fusion
vectors is described by Nilsson, B. et al. (Meth. Enzymol. 185:144-161
(1990)), herein incorporated by reference.
An especially preferred prokaryotic vector is one that, in lieu of
expressing the entire SpA protein, expresses only the IgG-binding domains
of the protein. Thus, such vectors express the protein fused to only a
portion of SpA. Most preferably, such vectors will be constructed such
that the expressed fusions will contain a cleavable site located between
the IgG-binding domains and the desired protein. Examples of such vectors
are described by Nilsson, B. et al. (Meth. Enzymol. 398:3-16 (1990)),
herein incorporated by reference. A particularly preferred example of such
a vector is pEZZ18 and its derivatives having cleavable sites (Nilsson, B.
et. al., Meth. Enzymol. 198:3-16 (1990)). The pEZZ18 vector contains a
lacZ gene sequence fused to the SpA IgG-binding domains. The lacZ
sequences are separated from the IgG domains by a multi-linker site, such
that a desired DNA fragment can be readily inserted into the vector. The
insertion of a desired sequence alters or obliterates the expression of
the LacZ protein, and hence can be detected using chromogenic substrates
(Nilsson, B. et al., Meth. Enzymol. 198:3-16 (1990)).
If desired, however, yeast and fungal vectors may be used. Examples of
suitable yeast vectors include the yeast 2-micron circle, the expression
plasmids YEP13, YCP and YRP, etc., or their derivatives. Such plasmids are
well known in the art (Botstein, D., et al., Miami Wntr. Symp. 19:265-274
(1982); Broach, J. R., In: The Molecular Biology of the Yeast
Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, J. R., Cell
28:203-204 (1982); Sambrook, J. et al., In: Molecular Cloning, A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. (1989)).
If alternatively desired, the inhibitors of the present invention may be
produced by expression in mammalian cells, using eukaryotic vectors
(especially, a eukaryotic viral or retroviral vector). Typically, such
vectors will be designed to include a prokaryotic replicon, and selectable
marker, such that the propagation of the vector in bacterial cells can be
readily accomplished. In order to replicate in mammalian cells, however,
the vectors will also contain a viral replicon, such as the replicon of
Epstein-Barr virus, bovine papilloma virus, parvovirus, adenovirus, or
papovavirus (i.e. SV40 or polyomavirus).
Plasmid vectors using papovavirus replicons ultimately kill their host
cells, and are thus preferred for transient expression. SV40-based vectors
that may be used include pMSG (Pharmacia), pSVT7, pMT2 (Kaufman, R. J.,
In: Genetic Engineering: Principles and Methods Vol. 9 (Setlow, J. K.,
Ed.) Plenum Publishing, N.Y. (1987)).
In contrast, vectors that employ the replicons of Epstein-Barr, or bovine
papilloma viruses do not generally cause cell death, and are thus suitable
for long term propagation. Examples of such vectors include BPV-1,
pBV-1MTHA, pHEBo, p205 (Shimuzu, Y. et al., Mol. Cell. Biol. 6:1074
(1986); Kioussis, D. et al. EMBO J. 6:355 (1987); Sambrook, J. et al.,
EMBO J. 4:91 (1985).
Sambrook, J. et al., herein incorporated by reference, provide a review of
the characteristics of mammalian vectors (In: Molecular Cloning, A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. (1989).
The use of recombinant DNA expression methods facilitates the isolation and
recognition of additional inhibitor molecules. In one embodiment, such
molecules can be subjected to random mutagenesis (see, for example,
Watson, J. D. et al., Molecular Biology of the Gene, Fourth Edition,
Benjamin/Cummings, Menlo Park, Calif. (1987)). Significantly, a random
mutagenesis approach requires no a priori information about the protein
encoded by the gene that is to be mutated. This approach has the advantage
that it assesses the desirability of a particular mutant on the basis of
its function, and thus does not require an understanding of how or why the
resultant mutant protein has adopted a particular conformation. Indeed,
the random mutation of target gene sequences has been one approach used to
obtain mutant proteins having desired characteristics (Leatherbarrow, R.
J. Prot. Eng. 1:7-16 (1986); Knowles, J. R., Science 236:1252-1258 (1987);
Shaw, W. V., Biochem. J. 246:1-17 (1987); Gerit, J. A. Chem. Rev.
87:1079-1105 (1987)).
The efficiency of the mutagenesis can be increased by recognizing that the
identity and location of critical or key amino acid residues has been
determined by virtue of the isolation and sequencing of the
above-described mutants. Thus, it is possible to use site-specific
mutagenesis techniques to selectively alter only those amino acids of the
protein that are believed to be important (Craik, C. S., Science
228:291-297 (1985); Cronin, C. S. et al., Biochem. 27:4572-4579 (1988);
Wilks, H. M. et al., Science 242:1541-1544 (1988)). Once the population of
mutants has been obtained, each mutant must be analyzed to determine
whether it fulfills the desired criteria. This analysis can be facilitated
through the use of a phage display protein ligand screening system
(Lowman, H. B. et al., Biochem. 30:10832-10838 (1991); Markland, W. et
al., Gene 109:13-19 (1991); Roberts, B. L. et al., Proc. Natl. Acad. Sci.
(U.S.A.) 89:2429-2433 (1992); Smith, G. P., Science 228:1315-1317 (1985);
Smith, R. P. et al., Science 248:1126-1128 (1990), all herein incorporated
by reference)). In general, this method involves expressing a fusion
protein in which the desired protein ligand is fused to the C-terminus of
a viral coat protein (such as the M13 Gene III coat protein, or a lambda
coat protein).
IV. Uses of the Inhibitors of the Present Invention
The present invention provides inhibitors of urokinase and similar
proteases. A preferred class of such inhibitors comprise "low molecular
weight" "proteinaceous" inhibitors. As used herein, an inhibitor is said
to be proteinaceous if it is a protein or polypeptide, or a derivative of
either. A proteinaceous inhibitor is said to be of "low molecular weight"
if the proteinaceous portion of the inhibitor has a molecular weight of
less than 20,000, and more preferably less than 15,000. Such desired
inhibitors are exemplified by the mutated OM3TKY molecules 88, 89, 90 and
91 described above.
Such inhibitors may be modified so as to contain or lack one, two, or
several amino acids from either the amino or carboxyl terminus. Similarly,
equivalent amino acids can be used anywhere in the protein in lieu of
those designated. As is known in the art, the amino acids may be present
in either their protected or unprotected forms, using appropriate amino or
carboxyl protecting groups. The molecule may have a free amine on its
amino terminus, or it may be prepared as an acid-addition salt, or
acetylated derivative. Examples of functionally active protein and peptide
analogues and functional derivatives, and methods for their preparation
are disclosed in U.S. Pat. Nos. 4,605,641 (Bolin et al.); 4,734,400 (Bolin
et al.); 4,822,774 (Ito et al.); 4,835,252 (Musso et al.); 4,939,224
(Musso et al.); 5,055,302 (Laties et al.), all herein incorporated by
reference).
As indicated above, urokinase plays an important role in a variety of
physiological processes in which the degradation of tissue occurs. Such
processes include, for example, the translation of tumor cells from the
site of a primary tumor to putative metastatic sites, the degradation of
mammary tissue following lactation, the process of ovulation, and the
process through which fertilized ova become implanted in the uterine wall
at the onset of pregnancy. Urokinase plays a role in effecting the release
of the oocyte from the follicle. Thus, urokinase inhibitors can prevent
pregnancy by impairing or preventing such release.
A use is said to be therapeutic if it alters a physiologic condition in a
desirable manner. The agents of the present invention may be used locally
or systemically to prevent or attenuate any "urokinase-dependent" process.
As used herein, a "urokinase-dependent" process is any physiological
process or reaction that involves and is facilitated by urokinase, or a
similar protease. The inhibitors of the present invention thus can be used
in cancer therapy to prevent or attenuate the metastatic potential of a
tumor. They can be used to prevent pregnancy in females by either
preventing ovulation, or by impairing the capacity of the fertilized ova
to implant itself into the uterine wall.
The agents of the present invention may be administered systemically. Such
administration is preferred, for example, in the treatment or prevention
of metastasis. Such administration may, for example, be by parenteral,
intravenous, or intranasal means. In some embodiments, such as in the
prevention of pregnancy, topical or local administration is preferred.
The agents of the present invention can be administered in either a
"prophylactic" or "therapeutic" manner. An agent that is administered in a
prophylactic manner is provided in advance of any identified need (for
example, as a contraceptive agent, or prior to the detection of a
potentially metastatic tumor). The administration of such a compound
serves to prevent or attenuate any urokinase-dependent process. In
contrast, an agent that is administered in a therapeutic manner is
provided at (or after) the onset of a symptom of a urokinase-dependent
process (such as, for example, the detection of a metastasis). The
administration of such a compound serves to attenuate the consequences of
the urokinase-dependent process (such as, for example, attenuating the
clinical significance, grade, size, number or consequence of the
metastasis).
The therapeutic agents of the present invention can be formulated according
to known methods used to prepare pharmaceutically useful compositions,
whereby these materials, or their functional derivatives, are combined in
admixture with a pharmaceutically acceptable carrier vehicle. Suitable
vehicles and their formulation, inclusive of other human proteins, e.g.,
human serum albumin, are described, for example, in Remington's
Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton Pa.
(1980)). In order to form a pharmaceutically acceptable composition
suitable for effective administration, such compositions will contain an
effective amount of such agents, together with a suitable amount of
carrier vehicle.
Additional pharmaceutical methods may be employed to control the duration
of action. Controlled release preparations may be achieved through the use
of polymers to complex or absorb the agents. The controlled delivery may
be exercised by selecting appropriate macromolecules (for example
polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate,
methylcellulose, carboxymethylcellulose, or protamine sulfate) and the
concentration of macromolecules as well as the methods of incorporation in
order to control release. Another possible method to control the duration
of action by controlled release preparations is to incorporate the agents
into particles of a polymeric material such as polyesters, polyamino
acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
Alternatively, instead of incorporating these agents into polymeric
particles, it is possible to entrap these materials in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly(methylmethacylate) microcapsules,
respectively, or in colloidal drug delivery systems, for example,
liposomes, albumin microspheres, microemulsions, nanoparticles, and
nanocapsules or in macroemulsions. Such techniques are disclosed in
Remington's Pharmaceutical Sciences (1980).
In one embodiment, the inhibitors of the present invention can be joined to
one or more additional moieties, including non-proteins or domains of
other proteins. Such joining may result from a conjugation of the
inhibitor molecules with the other molecule(s). Any of a variety of
methods can be used to effect such conjugation. For example, such
conjugation may be achieved by covalently modifying the inhibitor
molecules with a bivalent adduct or "crosslinking agent" (such as
glutaraldehyde, succinic anhydrides, etc.). Alternatively, such
conjugation may be achieved through the exploitation of ligand
interactions. For example, the affinity of biotin for avidin can be
exploited to fashion biotinylated derivatives that can be joined to one
another via avidin, avidin derivatives, or mercurated compounds.
In another embodiment, the joining of the inhibitors of the present
invention to domain(s) of other molecules, especially proteins, can be
accomplished using recombinant methods. In this embodiment, a nucleic acid
molecule is employed that, upon expression, yields an inhibitor molecule
of the present invention as a fusion protein, covalently bound (via a
peptide bond, or an amino acid or polypeptide bridge) to the domain(s) of
interest.
The inhibitors of the present invention can thus be joined to domain(s) of
other proteins, such as antibodies or receptor ligands or receptor
molecules, or to non-proteins. Preferred classes of protein molecules
include receptor ligands, and especially ligands that are capable of
specific or preferential binding to tumor cells. In this manner, the
conjugate protein can facilitate the transport of the inhibitor to the
site of the tumor. A particularly preferred ligand conjugate is the
cellular urokinase receptor ligand. The urokinase receptor is universally
present in and on tumor cells, especially on cells of the leading (or
metastatic) edge of a tumor mass. In view of such universal presence, the
use of a urokinase receptor ligand would permit a single molecule to be
used against all tumor types. In such an embodiment, the urokinase
inhibitor could preferentially bind to those cells that have arrayed the
urokinase receptor. In contrast, if one were to use tumor-specific
antigens as a target, it would be necessary to develop a library of tumor
antigen-specific antibodies.
In an alternative embodiment, "humanized" antibodies (i.e. non-human
antibodies which are non-immunogenic in a human) can be used as the
conjugate protein. Methods for producing such antibodies are well known
(Robinson, R. R. et al., International Patent Publication PCT/US86/02269;
Akira, K. et al., European Patent Application 184,187; Taniguchi, M.,
European Patent Application 171,496; Morrison, S. L. et al., European
Patent Application 173,494; Neuberger, M. S. et al., PCT Application WO
86/01533; Cabilly, S. et al., European Patent Application 125,023).
Whereas, in a preferred embodiment, the inhibitor molecules of the present
invention are provided per se to a recipient, the invention also
contemplates the administration of DNA or RNA molecules that encode and
express the inhibitor molecules as a method of gene therapy. In such an
embodiment of the present invention, DNA encoding an inhibitor is
introduced into the somatic cells of an animal (particularly mammals
including humans) in order to provide a treatment for cancer (i.e. "gene
therapy"). Most preferably, viral or retroviral vectors are employed for
this purpose.
The principles of gene therapy are disclosed by Oldham, R. K. (In:
Principles of Biotherapy, Raven Press, NY, 1987), and similar texts.
Disclosures of the methods and uses for gene therapy are provided by
Boggs, S. S. (Int. J. Cell Clon. 8:80-96 (1990)); Karson, E. M. (Biol.
Reprod. 42:39-49 (1990)); Ledley, F. D., In: Biotechnology, A
Comprehensive Treatise, volume 7B, Gene Technology, VCH Publishers, Inc.
NY, pp 399-458 (1989)); all of which references are incorporated herein by
reference.
Although, as indicated above, such gene therapy can be provided to a
recipient in order to treat (i.e. suppress, or attenuate) an
urokinase-dependent process, the principles of the present invention can
be used to provide a prophylactic gene therapy to individuals who, due to
inherited genetic mutations, somatic cell mutation, or pre-diagnosed
medical condition, have an enhanced probability of cancer, etc.
Having now generally described the invention, the same will be more readily
understood through reference to the following examples which are provided
by way of illustration, and are not intended to be limiting of the present
invention, unless specified.
EXAMPLE 1
Mutagenesis of Turkey Ovomucoid Third Domain Gene sequences that encode the
turkey ovomucoid third domain (OM3TKY) were mutagenized using plasmid,
pEZZ318TKYMET, which is a variant of the expression plasmid pEZZ318. The
plasmid contains an fl ori site, and an ampicillin resistance determinant.
The plasmid is capable of mediating the expression of OM3TKY as a fusion
protein, in which the OM3TKY sequences are fused to the C-terminus of two
tandem protein A domains. The expressed fusion protein has been engineered
to contain a cyanogen bromide cleavage site at the amino terminus of
OM3TKY. Thus, after synthesis, the fusion protein can be readily cleaved
to yield the desired OM3TKY sequences free of additional protein. The
recombinant protein expressed in E. coli, prior to chemical cleavage has
the sequence (SEQ ID NO:1):
__________________________________________________________________________
AAQHDEAVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSA
NLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQS
LKDDPSQSANLLAEAKKLNDAQAPKVDRKEAHFA MVDCSEYPKPACTLE
YRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC
__________________________________________________________________________
After cleavage (at the underlined methionine), the above sequence
comprising the turkey ovomucoid third domain is obtained (SEQ ID NO:2):
VDCSEYPKPACTLEYRPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC
In accordance with the above-described methods, site specific mutagenesis
was used to introduce mutations into a gene sequence that encodes the
OM3TKY protein. Four different oligonucleotides were used to mutagenize
the five codons around the coding region for the ovomucoid active site.
These oligonucleotides are:
1) The PAI-2 derived mutant, 88 (SEQ ID NO: 3): gtt gtc gga Gcc aca gag agg
ATG TCC CGT TCG TCC gca tgc agg
2) The PAI-1 derived mutant, 89 (SEQ ID NO:4): gtt gtc gga Gcc aca gag agg
TGC TGC CAT TCG TGC gca tgc agg
3) The Plasminogen derived mutant, 90 (SEQ ID NO:5:): gtt gtc gga Gcc aca
gag agg TCC AAC AAC TCG TCC gca tgc agg
4) The Nexin-1 derived mutant, 91 (SEQ ID NO:6): gtt gtc gga Gcc aca gag
agg TGC GGA GGA TCG TGC gca tgc agg
The regions of mutagenesis are shown in capital letters. At position 10
from the 5' end of each oligonucleotide, there is a mutation which
eliminates a unique BamHI site in pEZZ318TKY-MET, which facilitates
screening for mutants.
The oligonucleotides were kinased with T4 polynucleotide kinase, and then
single primer oligonucleotide-directed mutagenesis was done on single
stranded pEZZ318TKY-MET with a 15-fold molar excess of primer to template.
T4 ligase was added to the oligonucleotide-template reaction mixture
(including DTT and ATP), then dNTPs, and Klenow fragment (Amersham) were
added. Polymerization was allowed to proceed for 17 hours at 16.degree. C.
The reaction was transformed into competent mut1 cells, and then grown in
5 ml liquid culture LB media+ampicillin (100 .mu.g/ml) at 37.degree. C.
for 16 hours. Double-stranded DNA was prepared from the 5 ml growths by a
simplified procedure of the alkaline-lysis plasmid preparation method
(Sambrook, J., et al., Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989)).
After the plasmid DNA was re-dissolved in TE, 2 .mu.l was used in a BamHI
digestion. After two hours of digestion, 2 .mu.l of each BamHI reaction
was transformed into JM109 competent cells, plated onto LB agar+ampicillin
plates, and grown overnight at 37.degree. C. Colonies from these plates
were selected, grown and screened for BamHI resistance. The BamHI
digestion was carried out at 37.degree. C. for 90 minutes in a total
volume of 10 .mu.l, using 1 .mu.l BRL BamHI, and 1 .mu.l BRL React 3
Buffer(10.times.). Resistance to BamHI digestion was determined by running
the digests on a 1% agarose gel (agarose in Tris-Acetate-EDTA buffer). The
BamHI resistant samples were sequenced by the dideoxy chain termination
method. From the sequences, the desired four mutants were identified, and
used for expression of the mutant proteins.
The four inhibitor molecules thereby produced have the amino acid sequences
(the differences between the sequences are indicated by underscoring):
__________________________________________________________________________
88 (SEQ ID NO: 7):
VDCSEYPKPAC GRTGHPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC
89 (SEQ ID NO: 8):
VDCSEYPKPAC ARMAAPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC
90 (SEQ ID NO: 9):
VDCSEYPKPAC GRVVGPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC
91 (SEQ ID NO: 10):
VDCSEYPKPAC ARSSAPLCGSDNKTYGNKCNFCNAVVESNGTLTLSHFGKC
__________________________________________________________________________
EXAMPLE 2
Protein Expression and Purification
Protein expression of the mutagenized pEZZ-derived plasmids of Example 1
was carried out in JM109 cells, and the protein was harvested by an
osmotic shock procedure. A 100 ml culture of cells containing the plasmid
expressing the wild type or mutant gene was grown in 2xYT +ampicillin (100
.mu.g/ml) overnight at 37.degree. C. These cultures were centrifuged at
6000 rpm in an SS-34 rotor for ten minutes, and the supernatant was
discarded. The pellets were resuspended in 25 ml 2.times.YT each, and used
to inoculate 1 liter growths in 2.times.YT+ampicillin (100 .mu.g/ml),
which were grown for 4 hours at 37.degree. C. The one liter cultures were
then cooled down in an ice-water bath for 30 minutes. These growths were
then centrifuged at 8000 rpm in a GS-3 rotor for 15 minutes at 4.degree.
C. The supernatant was discarded, and the pellets frozen overnight at
-20.degree. C.
The frozen pellets were thawed, and resuspended in 25 ml cold sucrose
buffer (0.5M sucrose, 0.2M Tris-HCl (pH 8.2), 1 mM EDTA). After sitting
for 10 minutes in an ice water bath, 0.5 g of lysozyme was added to each
resuspension, immediately followed by 25 ml of ice cold distilled water.
This mixture was vortexed thoroughly, and then incubated in an ice-water
bath for 5 minutes. 1 ml of 1M MgSO.sub.4 was added to each lysate, and
the lysates were centrifuged in Oak Ridge tubes at 12000 rpm in an SS-34
rotor for 20 minutes. The supernatants from each tube were filtered
through a 0.45 micron filter, and if not immediately taken for further
purification, were frozen at -20.degree. C. until further use.
Affinity purification of proteins from each osmotic shock supernatant was
done on IgG-sepharose (Pharmacia) gels of 3-4 ml bed volume. The columns
were equilibrated with a 10.times.bed volume of TST (50 mM Tris-HCl (pH
7.4), 150 mM NaCl, 0.05% Tween 10) before loading the osmotic shock
supernatants. The columns were then washed with 300 ml of TST. The buffer
capacity of the column was then lowered by washing with 15 ml of 1 mM
ammonium acetate. The columns were eluted with 10 ml of 0.5M acetic acid
titrated to pH 3.2 with ammonium acetate. The eluates were collected in 10
ml of 1 M Tris-HCl (pH 8.0), and frozen at -70.degree. C. When completely
frozen, the samples were lyophilized. Lyophilized samples were
re-dissolved in 2 ml distilled water and frozen until further use.
SDS-PAGE was routinely used as a check on the expression and purification
procedures. A 12.5% polyacrylamide gel was used to resolve the proteins in
the IgG eluate, which was stained with Coomasie blue, and destained with
methanol/acetic acid. The major bands after purification corresponded in
apparent molecular weight to the Protein A-ovomucoid third domain fusion
protein and a breakdown product of two Protein A domains.
EXAMPLE 3
Quantification of Urokinase
In order to quantitate UK activity, a method of back titration from
quantitated trypsin was used with a calibrated chloromethyl ketone (CK)
inhibitor (Magnotti, R. A. et al., Anal. Biochem. 170:228-237 (1988)). The
difference in enzyme activity between inhibited and non-inhibited enzymes,
as determined by change in absorbance over time at 405 nm, was used to
calculate chloromethyl ketone concentration. From this calibrated CK
concentration, the concentration of urokinase was readily determined using
the formula:
[CA]=([Enzyme].times.(1-Fractional Residual Activity))
Fractional residual activity is the ratio of enzyme activity with CK in the
reaction to enzyme activity without CK in the reaction.
Titration of trypsin active sites was done using the burst titrant p -
nitrophenyl p - guanidinobenzoate (Chase T. et al., Biochem. Biophys. Res.
Commun. 29:508-614 (1967)). The reaction was performed in Buffer A (50 mM
Hepes, 20 mM CaCl.sub.2, 0.1% PEG 8000, at pH8.0 (Magnotti, R. A. et al.,
Anal. Biochem. 170:228-237 (1988)). Titrations were performed with a
trypsin solution containing 2 mg/ml Bovine Trypsin (Type III Sigma), using
50, 80 and 100 .mu.l of this stock solution. The bursts were read at 410
nm on a Uvikon 860 spectrophotometer in plastic cuvettes with a path
length of 1 cm. The trypsin solution used was determined to be
5.times.10.sup.-4 M.
Determination of urokinase active site concentration was done by a back
titration with an arginyl specific chloromethyl ketone (EGRCK:
L-glutamyl-glycyl-arginyl-chloromethyl ketone from Calbiochem) active
against UK and trypsin (Magnotti, R. A. et al., Anal. Biochem. 170:228-237
(1988)).
This chloromethyl ketone was first calibrated against a known concentration
of trypsin in Buffer A using the chromogenic substrate BAPNA (Sigma), and
determined to be at a concentration of 2.6.times.10.sup.-5 M. The UK
determination was done in Buffer C (50 mM Tris-HCl, 0.1M NaCl, 0.01% Tween
80, all at pH 8.0), using chromogenic substrate S-2288 (KabiGen) at 1 mM
concentration.
Protein samples were assayed to determine whether any of the mutants
exhibited UK inhibitory activity. "Abbokinase" (Urokinase for
Injection-Abbott Pharmaceuticals) was dissolved in distilled water and
assays were done in a solution containing 0.05% mannitol, 0.5% human
albumin, and 0.1% NaCl.
The results of this experiment are shown in FIG. 2. In the top panel of
FIG. 2, the activity (change in absorbance at 405 nm/minute) of
chymotrypsin is plotted. The lower panel of FIG. 2 provides a similar plot
of the activity of UK. Inhibitor concentration is expressed as moles of
inhibitor per moles of enzyme active site, and based on bichoncinninic
acid quantification of protein in the samples, and concentration of
wild-type inhibitor titrated against known chymotrypsin concentrations.
Substrate concentration in each reaction is 2 times K.sub.m.
The results (FIG. 2, top panel) show a loss of chymotrypsin inhibitory
function in the mutants. The results show that the mutants are able to
inhibit UK to a much greater degree than can wild type OM3TKY (FIG. 2,
lower panel).
EXAMPLE 4 l
Determination of K.sub.m for Mutant Inhibitors
Fractional enzyme activities for each inhibitor-UK interaction of the above
discussed inhibitors were determined by the assay method of Empie and
Laskowski (Empie, M. W. et al., Biochem. 21:2274-2284 (1982)), as adapted
from Green and Work (Green, N. M. et al., Biochem. J. 54:347-352 (1953)).
The concentration of UK used for these reactions was 35 nM. The reactions
were carried out in Buffer C. The concentration of the chromogenic
substrate used with UK in these assays (S-2288, KabiGen) was 50 .mu.M,
which is one tenth of the K.sub.m of the substrate for UK. K.sub.m was
determined by a Michaelis-Menten analysis, using a 100-fold range of
substrate concentration in Buffer C, and determined to be
4.times.10.sup.-4 M. K.sub.i values were calculated as the slope of a plot
of [I].sub.o /(1-a) vs. 1/a ("a" represents residual enzyme activity)
using a variation of the formulas derived by Henderson (Pratt, C. W. et
al., Biochem. 26:2855-2863 (1987)). Table 5 shows the K.sub.i values
obtained for the wild type OM3TKY, and five mutants. These five mutants
include the four detailed above(88, 89, 80, AND 91), and also a mutant
with arginine at the P1 site of OM3TKY (P1-Arg). As is shown in the Table,
wild type OM3TKY and the P1 Arg mutant displayed virtually no inhibitory
activity against UK. The values for PSTI, PAI-1, PAI-2, and Nexin-1 were
obtained from Turpeinen, U. et al. (Biochem. J. 254:911-914 (1988)),
Kruithof, C.K.O. et al. (Thromb.-Haemost. 55::65-68 (1986)), Christensen,
U. et al. (Thromb.-Haemost. 48:24-26 (1982)), and Stein, P. E. et al.
(Nature 347:99-102 (1990)), respectively. "ND" means not determined.
K.sub.i values for serpins are not functionally comparable to those of
reversible inhibitors.
TABLE 5
______________________________________
K.sub.i VALUES FOR INHIBITORS AGAINST UK
Inhibitor K.sub.i (nM)
______________________________________
WT-OM3TKY --
P1-Arg --
88 40
89 0.31
90 106
91 120
PSTI 300
PAI-1 ND
PAI-2 <<.01
Nexin-1 ND
______________________________________
The K.sub.i values in the above table support the validity of the initial
modelling and changes to the Kazal system, and indicate that modelling can
be used to yield urokinase inhibitors.
K.sub.i values for the wild-type and mutant inhibitors versus urokinase
were also determined by non-linear regression of the following equation:
##EQU1##
The non-linear regression was done using the program Enzfitter, by Robin
Leatherbarrow. E.sub.o represents a total enzyme concentration in the
reaction, I.sub.o represents total inhibitor concentration, E is the free
enzyme for a given inhibitor concentration and K.sub.i is the equilibrium
constant for the inhibitor. E and K.sub.i are the unknowns in the
equation, and are solved for during the regression analysis. Enzyme and
inhibitor concentrations used were determined as mentioned above. Using
this method, the K.sub.i values (in nM) reported in Table 6 were
determined.
TABLE 6
______________________________________
K.sub.i VALUES OBTAINED BY NON-LINEAR
REGRESSION FOR INHIBITORS AGAINST UK
Inhibitor K.sub.i (nM)
______________________________________
WT-OM3TKY 795.6 .+-. 260
P1-Arg 1788.2 .+-. 415
88 (PAI-2 derived Mutant)
67.6 .+-. 5.6
89 (PAI-1 derived Mutant)
35.9 .+-. 10.5
90 (Plasminogen derived Mutant)
51.3 .+-. 15.2
91 (Nexin derived Mutant)
505 .+-. 122
PSTI 300
______________________________________
The data for wild-type, and the "Arg18," "88," "89", "90," and "91" mutants
are shown graphically in FIGS. 3A-3F.
EXAMPLE 5
Differences in the P2-P4,' Regions of the Mutant Inhibitors
The differences between the P2-P4' regions in the above-described
inhibitors is shown in Table 7. The Table provides a sequence comparison
of the four engineered mutants, native Pancreatic Secretory Trypsin
Inhibitor (PSTI), and the natural inhibitors to UK. PSTI, a Kazal-type
inhibitor from humans, was included in Table 7 because it had been
reported that PSTI (identified as TATI), had some affinity to UK
(Turpeinen, U. et al. (Biochem, J. 254:911-914 (1988)).
TABLE 7
______________________________________
SEQUENCE COMPARISON OF P2-P4'
IN UK INHIBITORS
P2 P1 P1' P2' P3' P4' INHIBITOR
______________________________________
T L E Y R P OM3TKY
T R E Y R P P1 ARG
G R V V G P PLASMINOGEN
DERIVATIVE (90)
G R T G H P PAI-2 DERIVATIVE (88)
A R M A A P PAI-1 DERIVATIVE (89)
A R S S A P NEXIN-1 DERIVATIVE (91)
A R M A P E PAI-1
G R T G H G PAI-2
G R V V G H PLASMINOGEN
A R S S P P NEXIN-1
T K I Y N P PSTI
______________________________________
Unlike PSTI, all of the mutant inhibitors created by the methods of the
present invention, and all the natural inhibitors to UK, have a glycine or
alanine at P2, which might be adding to the flexibility of the P1 loop.
Such flexibility may be desirable for UK inhibition in that it appears
that more flexibility in the P1 loop of Kazal and Kunitz inhibitors might
work to increase their inhibitory effects against plasminogen activators.
Structural studies of ovalbumin, a non-inhibitory serpin, and the closest
sequence homologue to PAI-2, has shown a reactive center (P1 loop) in the
shape of an a-helix (Stein, P. E. et al. (Nature 347:99-102 (1990)). In
inhibitory serpins, recent studies have shown that this reactive center
loop is extremely mobile (Carrell, R. W. et al., Nature 353:576-578
(1991)), with the serpin having to be forced into an energetically
unstable position that exposes a large portion of P10-P14'. Increased
exposure to proteolysis is often viewed as a measure of increased
flexibility and movement in a protein segment (Onesti, S. et al., J.
Molec. Biol. 217:153-176 (1991)).
Studies on soybean-type trypsin inhibitors from Erythrina seeds shows that
it has affinity for t-PA (Onesti, S. et al., J. Molec. Biol. 217:153-176
(1991)). As mentioned above, only serpins had been known to be able to
bind and inhibit t-PA. When the structure of an inhibitor from E. caffra
was crystallographically determined (Onesti, S. et al., J. Molec. Biol.
217:153-176 (1991)), it showed a P1 loop with the same canonical form as
Kunitz and Kazal inhibitors for the backbone and C.beta. carbons. The
entire loop itself was more exposed from the surface, and more flexible,
as estimated from B-values, than the loops in Kazal inhibitors. Also, the
overall width of the P1 loop in the E. caffra inhibitor, as measured from
P4 to P3' C.alpha. is 3 to 6.5 .ANG. narrower than in Kunitz and Kazal
inhibitors (Onesti, S. et al., J. Molec. Biol. 217:153-176 (1991)).
Increasing flexibility between the P3 and P6' cystines might enable the
Kazal derived inhibitors to bind plasminogen activators better than they
currently do.
EXAMPLE 6
Contact Determinants
The inhibitory capacity of the molecules of the present invention may
result either from the introduction of residues in the OM3TKY that make
better contacts with UK than the wild type OM3TKY does, or the removal of
residues that formed bad contacts between wild type OM3TKY and UK. The
fact that the P1 change to arginine did not increase affinity indicates
that other residues, besides the residue at P1, are also determinants of
binding. The presence of a bulky .beta.-branched residue, Ile at P1' and
an aromatic Tyr, at P2' in PSTI would indicate that the size of these
residues and simple steric contacts between these residues and urokinase
are not a major determinant of specificity. The nature of the contacts,
such as possible charge interactions and hydrogen bonds, may be the
determining factors.
Also of interest is a report of the basis of trypsin-like serine protease
specificity for natural substrates (Hedstrom, L. et al., Science
255:1249-1253 (1992)). This report suggested that while the S1 residue of
a substrate contains the specificity for the cleavage site, other sections
of the protease, outside the active site pocket, help to determine which
proteins are chosen as substrates. These sections are residues 184-189 and
221-225 of chymotrypsin and represent loops that border the sequences that
make up the chymotrypsin hydrophobic binding pocket (Hedstrom, L. et al.,
Science 255:1249-1253 (1992)). In the modelling of the present invention,
these residues did not appear to come into proximity with either BPTI or
OM3TKY inhibitors.
EXAMPLE 7
Ligand Searching by Phage Display
Further mutations and refinements to the above-described mutants can be
accomplished by creating a library of inhibitors that bind UK, and which
would therefore aid in identifying the permissible contact residues.
In order to generate a library of inhibitors with affinity, and possibly
specificity, for UK more rapidly, the method of protein ligand screening
known as a phage display system can be employed. In general, this method
involves expressing a fusion protein in which the desired protein ligand
is fused to the C-terminal domains of the M13 Gene III coat protein
(Lowman, H. B. et al., Biochem. 30:10832-10838 (1991); Markland, W. et
al., Gene 109:13-19 (1991); Roberts, B. L. et al., Proc. Natl. Acad. Sci.
(U.S.A.) 89:2429-2433 (1992); Smith, G. P., Science 228:1315-1317 (1985);
Smith, R. P. et al., Science 248:1126-1128 (1990), all herein incorporated
by reference). A fusion of the Pancreatic Secretory Trypsin Inhibitor
(PSTI) to the C-terminal domain of M13 Gene III can be made. Expression of
mutants of this fusion coat protein on the surface of M13 phages results
in phages that can be passed through a urokinase affinity column, and
allows for purification of the DNA packaged within the phages. This DNA
will code for the mutants with affinity for UK, and can subsequently be
amplified and sequenced.
In one embodiment, a plasmid, to be called pTACZZG3, can be constructed
which has tetracycline and ampicillin resistance, fl origin, the tac
inducible promoter, the signal sequence and two protein A domains from
pEZZ318, and the C-terminal portion of M13 Gene III. This fragment should
contain the M13 Gene III region coding from residue 197 of the Gene III
product to the AluI site at nucleotide position 2964 (GCG numbering) of
M13.
An insert coding for PSTI is constructed from four synthetic
oligonucleotides (Table 4) and ligated into pTACZZG3 between the EcoRI and
SstI sites. This will direct the synthetic PSTI gene into the proper
orientation, and allow for proper peptide linker regions to be in the
expressed polypeptide, and the insertion of an amber stop codon following
the coding sequences of the PSTI gene. This synthetic gene sequence has
three unique restriction sites, which aid in the insertion of randomized
DNA cassettes at the intended sites of mutagenesis in the inhibitor.
Cassette mutagenesis is thus conducted on this construct by methods
analogous to those described by Lowman, H. B. (Biochem. 30:10832-10838
(1991)). Screening of PSTI variants is done by M13K07 assisted growth of
phage with the PSTI-Gene III fusion expressed on the phage surface
(Lowman, H. B. et al., Biochem. 30:10832-10838 (1991)). These altered
phages are bound to urokinase attached to a solid phase (for example,
beads) to select for PSTI variants on the phage with increased affinity to
UK. These selected phage can be grown and sequenced, and also
alternatively expressed in a non-amber suppressor strain to obtain a
Protein A/PSTI fusion protein without the C-terminal portion of Gene III
expressed (Lowman, H. B. et al., Biochem. 30:10832-10838 (1991)). Other
methods of combinatorial library screening, which will be apparent to
those skilled in the art, can also be used to isolate PSTI-like or other
Kazal-type inhibitors having increased affinity to urokinase. For example,
the a synthetic approach to combinatorial cassette nutagenesis (or "CCM")
may be used (Hermes, J. D. et al., Proc. Natl. Acad. Sci. (U.S.A.)
87:696-700 (1990); Lim, W. A. et al. Nature 339:31-36 (1989);
Reidhaarolson, J. F. et al., Science 241:53-57 (1988); Oliphant, A. R., et
al., Gene 44:177-183 (1986), all herein incorporated by reference).
Alternatively, methods of random mutagenesis (Leatherbarrow, R. J. Prot.
Eng. 1:7-16 (1986); Knowles, J. R., Science 236:1252-1258 (1987); Shaw, W.
V., Biochem. J. 246:1-17 (1987); Gerit, J. A. Chem. Rev. 87:1079-1105
(1987)) or site-directed mutagenesis (Craik, C. S., Science 228:291-297
(1985); Cronin, C. S. et al., Biochem. 27:4572-4579 (1988); Wilks, H. M.
et al., Science 242:1541-1544 (1988))) may be employed in order to produce
the desired variant proteins and polypeptide mimetics.
While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations,
uses, or adaptations of the invention following, in general, the
principles of the invention and including such departures from the present
disclosure as come within known or customary practice within the art to
which the invention pertains and as may be applied to the essential
features hereinbefore set forth and as follows in the scope of the
appended claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 10
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 184 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: STAPHYLOCCOCAL PROTEIN A - TURKEY OVOMUCOID
DOMAIN FUSION
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AlaAlaGlnHisAspGluAlaValAspAsnLysPheAsnLysGluGln
151015
GlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuAsnGluGlu
202530
GlnArgAsnAlaPheIleGlnSerLeuLysAspAspProSerGlnSer
354045
AlaAsnLeuLeuAlaGluAlaLysLysLeuAsnAspAlaGlnAlaPro
505560
LysValAspAsnLysPheAsnLysGluGlnGlnAsnAlaPheTyrGlu
65707580
IleLeuHisLeuProAsnLeuAsnGluGluGlnArgAsnAlaPheIle
859095
GlnSerLeuLysAspAspProSerGlnSerAlaAsnLeuLeuAlaGlu
100105110
AlaLysLysLeuAsnAspAlaGlnAlaProLysValAspArgLysGlu
115120125
AlaHisPheAlaMetValAspCysSerGluTyrProLysProAlaCys
130135140
ThrLeuGluTyrArgProLeuCysGlySerAspAsnLysThrTyrGly
145150155160
AsnLysCysAsnPheCysAsnAlaValValGluSerAsnGlyThrLeu
165170175
ThrLeuSerHisPheGlyLysCys
180
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: TURKEY OVOMUCOID DOMAIN 3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ValAspCysSerGluTyrProLysProAlaCysThrLeuGluTyrArg
151015
ProLeuCysGlySerAspAsnLysThrTyrGlyAsnLysCysAsnPhe
202530
CysAsnAlaValValGluSerAsnGlyThrLeuThrLeuSerHisPhe
354045
GlyLysCys
50
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: 88 OLIGONUCLEOTIDE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GTTGTCGGAGCCACAGAGAGGATGTCCCGTTCGTCCGCATGCAGG45
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: 89 OLIGONUCLEOTIDE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GTTGTCGGAGCCACAGAGAGGTGCTGCCATTCGTGCGCATGCAGG45
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: 90 OLIGONUCLEOTIDE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GTTGTCGGAGCCACAGAGAGGTCCAACAACTCGTCCGCATGCAGG45
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: 91 OLIGONUCLEOTIDE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GTTGTCGGAGCCACAGAGAGGTGCGGAGGATCGTGCGCATGCAGG45
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vii) IMMEDIATE SOURCE:
(B) CLONE: OM3TKY clone 88 inhibitor
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ValAspCysSerGluTyrProLysProAlaCysGlyArgThrGlyHis
151015
ProLeuCysGlySerAspAsnLysThrTyrGlyAsnLysCysAsnPhe
202530
CysAsnAlaValValGluSerAsnGlyThrLeuThrLeuSerHisPhe
354045
GlyLysCys
50
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vii) IMMEDIATE SOURCE:
(B) CLONE: OM3TKY clone 89 inhibitor
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ValAspCysSerGluTyrProLysProAlaCysAlaArgMetAlaAla
151015
ProLeuCysGlySerAspAsnLysThrTyrGlyAsnLysCysAsnPhe
202530
CysAsnAlaValValGluSerAsnGlyThrLeuThrLeuSerHisPhe
354045
GlyLysCys
50
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vii) IMMEDIATE SOURCE:
(B) CLONE: OM3TKY clone 90 inhibitor
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ValAspCysSerGluTyrProLysProAlaCysGlyArgValValGly
151015
ProLeuCysGlySerAspAsnLysThrTyrGlyAsnLysCysAsnPhe
202530
CysAsnAlaValValGluSerAsnGlyThrLeuThrLeuSerHisPhe
354045
GlyLysCys
50
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vii) IMMEDIATE SOURCE:
(B) CLONE: OM3TKY clone 91 inhibitor
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
ValAspCysSerGluTyrProLysProAlaCysAlaArgSerSerAla
151015
ProLeuCysGlySerAspAsnLysThrTyrGlyAsnLysCysAsnPhe
202530
CysAsnAlaValValGluSerAsnGlyThrLeuThrLeuSerHisPhe
354045
GlyLysCys
50
__________________________________________________________________________
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